RESUMEN
To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 ( PfCDPK1) as a strategy to disrupt J domain binding and inhibit PfCDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant PfCDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of PfCDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.
Asunto(s)
Malaria Falciparum/parasitología , Péptidos/química , Péptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Eritrocitos/parasitología , Humanos , Cinética , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Dominios Proteicos , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 µM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.
Asunto(s)
Antimaláricos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/metabolismo , Antimaláricos/química , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Péptidos/química , Proteínas Protozoarias/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteína Quinasa Tipo II Dependiente de AMP Cíclico/antagonistas & inhibidores , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/antagonistas & inhibidores , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Subunidades de Proteína/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/química , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/metabolismo , Proteína Quinasa Tipo II Dependiente de AMP Cíclico/química , Proteína Quinasa Tipo II Dependiente de AMP Cíclico/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Péptidos/farmacología , Fosforilación , Unión Proteica/efectos de los fármacos , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells.